Many internal organs, such as the kidney, consist of hollow tubules and spheres lined by a layer of polarized epithelial cells. These cells have an apical plasma membrane domain facing the central lumen and a basolateral plasma membrane facing the underlying basement membrane. These two plasma membrane domains have completely different protein and lipid compositions, and trafficking of proteins and lipids to these distinct membrane surfaces is vital. We have found that the signaling lipid phosphatidyl inositol 3,4,5- trisphosphate (PIP3) is found only at the basolateral plasma membrane and is a key determinant of the identity and formation of this surface. In contrast phosphatidyl inositol 4,5-bisphosphate (PIP2) is concentrated at the apical plasma membrane, where it is a main determinant of this surface. We will test the hypothesis that PIP2 and PIP3 control the development of epithelial polarity, using live cell imaging. We will test the hypothesis that PIP3 is synthesized at the basolateral plasma membrane by a specific isoform(s) of phosphatidyl inositiol 3- kinase. We will test the respective roles of the lipid phosphatases PTEN and SHIP1/2 in preventing PIP3 from accumulating at the apical plasma membrane. We will test the hypothesis that gp135/podocalyxin and proteins with which it interacts are directly involved in formation of the apical surface. Gp135 is a negatively charged, transmembrane sialomucin, which binds via a PDZ motif at its C-terminus to the scaffolding protein NHERF1. We will mislocalize gp135 to the basolateral surface and see if NHERF1 and other proteins follow. Much of the PIP2 at the plasma membrane is synthesized by phosphatidyl inositol 4- phosphate 5-kinase (PI5K). The isoform PI5K1beta is found at the apical plasma membrane and interacts with NHERF1. We will test the involvement of PI5K1beta (as well as the alpha and gamma isoforms) in formation of the apical plasma membrane. We will also test if PTEN is recruited to the apical plasma membrane by its interaction with NHERF. Together, these experiments will help us understand the molecular mechanism of apical plasma membrane and lumen formation.